1. Field of the Invention
The present invention relates generally to the fields of biochemistry, cell biology, and oncology. More specifically, it concerns methods for modulating RAD51 protein activity in a cell.
2. Description of Related Art
Homologous recombination (HR) has multiple roles in DNA repair including the repair of DNA double-strand breaks (DSBs) and recovery from the replication blocking lesions formed by DNA cross-linking agents. HR repairs DSBs by locating a homologous stretch of DNA and replicating the missing genetic information from the homologous template. In contrast to repair by non-homologous end joining (NHEJ), DSB repair by HR generally occurs without mutations (Thompson and Schild, 2001). Numerous studies have also shown HR to be critically important in the maintenance of genomic stability (Thompson and Schild, 2001; Godthelp et al., 2002; Tebbs et al., 1995; Takata et al., 2001; Liu et al., 2002; Cui et al., 1999; Thompson and Schild, 1999). The proposed mechanism for this pathway begins with 5′ to 3′ nuclease activity at the DSB, resulting in a 3′ single stranded tail (FIG. 1). The tail is coated by Replication Protein A (RPA), which is subsequently replaced by a helical filament of RAD51 protein. This displacement of RPA by RAD51 appears to be mediated by a number of protein complexes, which include RAD52 and a family of RAD51 paralog proteins (Thompson and Schild, 2001; Gasior et al., 2001). These RAD51 filaments can be microscopically visualized with fluorescent antibodies, and they appear as sub-nuclear foci (Bishop et al., 1998). The RAD51 coated 3′ tail then invades a double stranded stretch of homologous template DNA. The genetic code is essentially copied from this template by polymerase activity and branch migration, in a structure termed the Holliday junction.
In addition to RAD51, repair via HR requires five RAD51 paralog proteins. The paralogs form two complexes in solution, a XRCC3/RAD51C heterodimer and a RAD51B/RAD51C/RAD51/XRCC2 heterotetramer (Liu et al., 2002; Masson et al., 2001a; Masson et al., 2001b; Wiese et al., 2002). Mutation of any one of the five paralog genes prevents subnuclear assembly of recombinase at damaged sites and reduces the cell's ability to perform HR repair (Godthelp et al., 2002; Tebbs et al., 1995; Takata et al., 2001; Bishop et al., 1998; Fuller and Painter, 1988; Caldecott and Jeggo, 1991; Liu et al., 1998). These paralogs are thought to serve as assembly ‘mediators’ for RAD51. RPA can inhibit assembly of RAD51 recombinase at sites of damage, and mediator proteins are thought to help overcome this inhibition. Studies have demonstrated a sensitization to certain DNA damaging therapies associated with cellular defects in proteins that promote HR DNA repair. This sensitization is particularly dramatic for DNA cross-linking chemotherapeutic drugs (30-100 times) and ionizing radiation (3-5 times) (Godthelp et al., 2002; Tebbs et al., 1995; Takata et al., 2001; Liu et al., 1998).
Several groups have recently demonstrated that HR can be partially inhibited in order to sensitize cells to DNA damaging therapies. Inhibition of XRCC3 (a RAD51 paralog protein), has been demonstrated using a synthetic peptide corresponding to another paralog protein. This peptide sensitized Chinese Hamster Ovary (CHO) cells to cisplatin and inhibited the formation of sub-nuclear RAD51 foci in response to DNA damage (Connell et al., 2004). Other researchers have inhibited the expression of the RAD51 protein itself (Russell et al., 2003; Hansen et al., 2003; Ohnishi et al., 1998; Ito et al., 2005; Collis et al., 2001) or blocked its function by over-expressing a dominant negative BRC peptide fragment derived from BRCA2 (Chen et al., 1999).
In view of the connection between increased sensitivity to certain DNA damaging therapies and cellular defects in HR DNA repair-related proteins, methods and compounds that provide for selective manipulation of this balance are desirable.